Abstract Nuclear factor-[unreadable]B (NF-[unreadable]B) is a transcription factor that plays many essential roles in immune responses in vivo, from contributing to the formation of lymph nodes and Peyer's patches to allowing generation and maturation of dendritic cells and controlling development and function of several T cell subsets. Several current immunosuppressive drugs to prevent solid organ transplant rejection, as well as many more in development, inhibit NF-[unreadable]B as part of their mechanism of action. However, NF-[unreadable]B is a ubiquitous transcription factor the inhibition of which can cause many severe side effects in different organ systems. In our previous funding period we showed that T cell-restricted inhibition of NF-[unreadable]B, using transgenic mice that express an NF-[unreadable]B super-repressor selectively in T cells (I[unreadable]B[unreadable][unreadable]N-Tg mice), results in permanent acceptance of fully allogeneic cardiac allografts and development of donor-specific tolerance via apoptosis of alloreactive T cells. In these mice, T cell-NF-[unreadable]B activity is inhibited downstream of several receptors, including the TCR, Toll-like receptors (TLRs) and tumor necrosis factor receptor (TNFR) family members. To determine whether selective inhibition of TCR-NF-[unreadable]B is sufficient for preventing allograft rejection, we have obtained CARMA1-KO mice that lack an adaptor molecule required for selectively linking the TCR and BCR to NF-[unreadable]B. Our new results indicate that CARMA1-KO mice permanently accept fully allogeneic cardiac allografts, positioning the CARMA1-NF-[unreadable]B axis as a very promising therapeutic target for clinical transplantation. Importantly, mice deficient in CARMA1 are an invaluable tool to understand the consequences of inhibition of this pathway on the development and differentiation of T cell subsets. We anticipate that drugs that would target NF-[unreadable]B selectively in T cells rather than in all cell types may have fewer side effects than current immunosuppressive therapies while having the potential to induce transplantation tolerance. However, before developing such inhibitors, it is important to understand fully the function of TCR-CARMA1-NF-[unreadable]B activity. Our current results indicate that targeting CARMA1 abolishes the thymic development of natural regulatory T cells (nTregs). Conversely, lack of CARMA1 promotes the differentiation of induced regulatory T cells (iTregs), underscoring CARMA1 as the only molecule described to date with opposite functions in the generation of nTregs versus iTregs. Further supporting reciprocal requirements for iTreg and Th17 differentiation, our preliminary results indicate that absence of CARMA1 prevents Th17 differentiation. These results reveal novel functions of the TCR-CARMA1-NF-[unreadable]B pathway that need to be substantiated mechanistically. The main goal of the current proposal is to define the precise role of CARMA1 and TCR-driven NF-[unreadable]B in the development/differentiation of T cell subsets in vitro and in transplantation models in vivo. The long-term underlying hypothesis for this project is that inhibition of TCR-driven NF-[unreadable]B may be a valuable approach to induce transplantation tolerance in vivo. Completion of our goals will bring us closer to developing drugs that can target this pathway and be able to test their potential to induce donor- specific tolerance in the clinic. Specific Aim 1. To determine the mechanism by which CARMA1 regulates the thymic development of nTregs. Specific Aim 2. To delineate the mechanism by which CARMA1-NF-[unreadable]B reciprocally regulates iTreg versus Th17 differentiation.